1. Field
The present invention relates to an improved production method for factor VIII and its derivatives. The method relates generally to vector construction, transfection, and selection of cell lines with enhanced productivity under protein-free conditions. In particular, this invention relates to a process for preparing a protein with factor VIII procoagulant activity on an industrial scale.
2. Background
Human factor VIII is a trace plasma glycoprotein involved as a cofactor in the activation of factor X and factor IXa. Inherited deficiency of factor VIII results in the X-linked bleeding disorder hemophilia A which can be treated successfully with purified factor VIII. The replacement therapy of hemophilia A has evolved from the use of plasma-derived factor VIII to the use of recombinant factor VIII obtained by cloning and expressing the factor VIII cDNA in mammalian cells. (Wood et al., 1984, Nature 312: 330).
Factor VIII has a domain organization of A1-A2-B-A3-C1-C2 and is synthesized as a single chain polypeptide of 2351 amino acids, from which a 19-amino acid signal peptide is cleaved upon translocation into the lumen of the endoplasmic reticulum. Due to the fact that factor VIII is heavily glycosylated, high-level expression ( greater than 0.2 pg/c/d) of factor VIII has been difficult to achieve (Lind et al., 1995, Eur J Biochem. 232: 19-27; Kaufman et al., 1989, Mol Cell Biol. 9: 1233-1242). Expression of factor VIII in mammalian cells is typically 2-3 orders of magnitude lower than that observed with other genes using similar vectors and approaches. The productivity of production cell lines for factor VIII has been in the range of 0.5-1 xcexcU/c/d (0.1-0.2 pg/c/d).
It has been demonstrated that the B-domain of factor VIII is dispensable for procoagulant activity. Using truncated variants of factor VIII, improved expression of factor VIII in mammalian cells has been reported by various groups (Lind et al., 1995, Eur J Biochem 232: 19-27; Tajima et al., 1990, Proc 6th Int Symp H.T. p.51-63; U.S. Pat. No. 5,661,008 to Almstedt, 1997). However, the expression level of the factor VIII variants remained below 1 pg/c/d from a stable cell clone.
We have now discovered (i) a method which derives cell lines with extremely high productivity of proteins having factor VIII procoagulant activity, and (ii) a plasma protein-free production process for proteins having factor VIII procoagulant activity.
A process for the production of proteins having factor VIII procoagulant activity at the industrial scale is disclosed. Using a newly created cell host, cell clones with specific productivities in the range of 2-4 pg/cell/day (10-20 xcexcU/c/d) were derived. Under serum-free conditions, one clone has sustained a daily productivity of 2-4 pg/c/d. Clones with this high level of productivity are able to produce 3-4 million units per day in a 15-liter perfusion fermenter. One unit of factor VIII activity is by definition the activity present in one milliliter of plasma. One pg of factor VIII is generally equivalent to about 5 xcexcU of FVIII activity.
As used herein, a protein having factor VIII procoagulant activity is a protein which causes the activation of Factor X in an in vitro or in vivo model system. As non-limiting examples, this definition includes full length recombinant human factor VIII and the B domain deleted factor VIII whose sequence is described in FIG. 1.
A high level of expression of a protein having factor VIII procoagulant activity means at least about 2 xcexcU/c/d, or more preferably at least about 4 xcexcU/c/d, or most preferably at least about 5 xcexcU/c/d, of factor VIII activity if grown in plasma derived protein-free medium, or at least about 4 xcexcU/c/d, or more preferably at least about 8 xcexcU/c/d, or most preferably at least about 10 xcexcU/c/d, of factor VIII activity if grown in medium supplemented with plasma derived protein. When the protein expressed is BDD-FVIII, cell lines having specific productivities up to about 15 xcexcU/c/d, more preferably up to about 20 xcexcU/c/d may be obtained by the method described herein.
As used herein to describe the origin of cell lines, xe2x80x9cderived fromxe2x80x9d is intended to include, but not be limited to, normal mitotic cell division and processes such as transfections, cell fusions, or other genetic engineering techniques used to alter cells or produce cells with new properties.